Niaz Banaei

Associate Professor of Pathology and of Medicine (Infectious Diseases) at the Stanford University Medical Center

Bio

Bio

Niaz Banaei received his medical education from Stanford University and completed residency training in laboratory medicine at the University of California, San Francisco. He then completed a postdoctoral fellowship in infectious diseases at the New York University. He is currently an Associate Professor of Pathology and Medicine (Division of Infectious Diseases & Geographic Medicine) at Stanford University and is the Medical Director of the Clinical Microbiology Laboratory at Stanford Health Care. In addition, he is the Director of Stanford Clinical Microbiology Fellowship. He serves on the board of advisors for the Center for Disease Control Tuberculosis Epidemiologic Studies Consortium II (TBESC). His research interests include (1) development, assessment, and improvement of novel infectious diseases diagnostics, (2) enhancing the quality of C. difficile diagnostic results, and (3) characterization of M. tuberculosis virulence determinants. He was the recipient of Kenneth L. Vosti Infectious Diseases and Stanford University clinical pathology junior faculty teaching awards.

Abstract

The Clostridium difficile rate in symptomatic patients represents both those with C. difficile infection (CDI) and those with colonization. To predict the extent of CDI overdiagnosis, we compared the asymptomatic colonization rate to the symptomatic positivity rate in hospitalized patients using nucleic acid testing.Between July 2014 and April 2015, formed stool samples were collected from asymptomatic patients after admission to 3 hospital wards at the Stanford Hospital. Stool samples from symptomatic patients with suspected CDI in the same wards were collected for testing per provider order. The GeneXpert C. difficile tcdB polymerase chain reaction (PCR) assay (Cepheid, Sunnyvale, CA, USA) was performed on all stool samples and PCR cycle threshold was used as a measure of genomic equivalents. Chart review was performed to obtain clinical history and medication exposure.We found an asymptomatic C. difficile carriage rate of 11.8% (43/365) (95% confidence interval [CI], 8.5-15.1%) and a positivity rate in symptomatic patients of 15.4% (54/351) (95% CI, 11.6-19.2%; P=0.19). The median PCR cycle thresholds was not significantly different between asymptomatic carriers and symptomatic positives (29.5 versus 27.3; P=0.07). Among asymptomatic patients, 11.6% (5/43) of carriers and 8.4% (27/322; P=0.56) of noncarriers subsequently became symptomatic CDI suspects within the same hospitalization. Single and multivariate analysis did not identify any demographic or clinical factors as being significantly associated with C. difficile carriage.Asymptomatic C. difficile carriage rate was similar to symptomatic positivity rate. This suggests the majority of PCR-positive results in symptomatic patients are likely due to C. difficile colonization. Disease-specific biomarkers are needed to accurately diagnose patients with C. difficile disease.

Abstract

Timely and accurate identification and determination of the antimicrobial susceptibility of uropathogens is central to the management of UTIs. Urine dipsticks are fast and amenable to point-of-care testing, but do not have adequate diagnostic accuracy or provide microbiological diagnosis. Urine culture with antimicrobial susceptibility testing takes 2-3 days and requires a clinical laboratory. The common use of empirical antibiotics has contributed to the rise of multidrug-resistant organisms, reducing treatment options and increasing costs. In addition to improved antimicrobial stewardship and the development of new antimicrobials, novel diagnostics are needed for timely microbial identification and determination of antimicrobial susceptibilities. New diagnostic platforms, including nucleic acid tests and mass spectrometry, have been approved for clinical use and have improved the speed and accuracy of pathogen identification from primary cultures. Optimization for direct urine testing would reduce the time to diagnosis, yet these technologies do not provide comprehensive information on antimicrobial susceptibility. Emerging technologies including biosensors, microfluidics, and other integrated platforms could improve UTI diagnosis via direct pathogen detection from urine samples, rapid antimicrobial susceptibility testing, and point-of-care testing. Successful development and implementation of these technologies has the potential to usher in an era of precision medicine to improve patient care and public health.

Abstract

Health care-onset health care facility-associated Clostridium difficile infection (HO-CDI) is overdiagnosed for several reasons, including the high prevalence of C. difficile colonization and the inability of hospitals to limit testing to patients with clinically significant diarrhea. We conducted a quasiexperimental study from 22 June 2015 to 30 June 2016 on consecutive inpatients with C. difficile test orders at an academic hospital. Real-time electronic patient data tracking was used by the laboratory to enforce testing criteria (defined as the presence of diarrhea [≥3 unformed stools in 24 h] and absence of laxative intake in the prior 48 h). Outcome measures included C. difficile test utilization, HO-CDI incidence, oral vancomycin utilization, and clinical complications. During the intervention, 7.1% (164) and 9.1% (211) of 2,321 C. difficile test orders were canceled due to absence of diarrhea and receipt of laxative therapy, respectively. C. difficile test utilization decreased upon implementation from an average of 208.8 tests to 143.0 tests per 10,000 patient-days (P < 0.001). HO-CDI incidence rate decreased from an average of 13.0 cases to 9.7 cases per 10,000 patient-days (P = 0.008). Oral vancomycin days of therapy decreased from an average of 13.8 days to 9.4 days per 1,000 patient-days (P = 0.009). Clinical complication rates were not significantly different in patients with 375 canceled orders compared with 869 episodes with diarrhea but negative C. difficile results. Real-time electronic clinical data tracking is an effective tool for verification of C. difficile clinical testing criteria and safe reduction of inflated HO-CDI rates.

Abstract

FilmArray GI Panel (BioFire Diagnostics, Salt Lake City, UT) is a simple, sample-to-answer, on-demand, multiplex, nucleic acid amplification test for syndromic diagnosis of infectious gastroenteritis. The aim of this study was to measure the yield of follow-up testing with FilmArray GI Panel within 4 weeks of an initial test. Consecutive adult and pediatric patients tested at an academic institution between August 2015 and June 2016 were included in this study. Of 145 follow-up tests in 106 unique patients with an initial negative result, 134 (92.4%) tests and 98 (92.5%) patients remained negative upon follow-up testing. Excluding targets that are not reported at our institution (Clostridium difficile, enteroaggregative Escherichia coli, enteropathogenic E. coli, and enterotoxigenic E. coli), 137 (94.5%) follow-up tests and 101 (95.3%) patients remained negative. Weekly conversion rates were not significantly different across the 4-week follow-up interval. No epidemiological or clinical factors were significantly associated with a negative to positive conversion. Of 80 follow-up tests in patients with an initial positive result, 43 (53.8%) remained positive for the same target, 34 (42.5%) were negative, and 3 were positive for a different target (3.8%). Follow-up testing with FilmArray GI Panel within 4 weeks of a negative result rarely changed the initial result, and the follow-up test reverted to negative less than half the time after an initial positive result. In the absence of clinical or epidemiological evidence for a new infection, follow-up testing should be limited and FilmArray GI Panel should not be used as a test of cure.

Abstract

Pseudomonas aeruginosa and Aspergillus fumigatus are the leading bacterial and fungal pathogens in cystic fibrosis (CF). We have shown that Af biofilms are susceptible to Pseudomonas, particularly CF phenotypes. Those studies were performed with a reference virulent non-CF Aspergillus. Pseudomonas resident in CF airways undergo profound genetic and phenotypic adaptations to the abnormal environment. Studies have also indicated Aspergillus from CF patients have unexpected profiles of antifungal susceptibility. This would suggest that Aspergillus isolates from CF patients may be different or altered from other clinical isolates. It is important to know whether Aspergillus may also be altered, as a result of that CF environment, in susceptibility to Pseudomonas. CF Aspergillus proved not different in that susceptibility.

Abstract

Understanding the contribution of relapse and reinfection to recurrent Clostridium difficile infection (CDI) has implications for therapy and infection prevention, respectively. We used whole genome sequencing to determine the relation of C. difficile strains isolated from patients with recurrent CDI at an academic medical center in the United States. Thirty-five toxigenic C. difficile isolates from 16 patients with 19 recurrent CDI episodes with median time of 53.5days (range, 13-362) between episodes were whole genome sequenced on the Illumina MiSeq platform. In 84% (16) of recurrences, the cause of recurrence was relapse with prior strain of C. difficile. In 16% (3) of recurrent episodes, reinfection with a new strain of C. difficile was the cause. In conclusion, the majority of CDI recurrences at our institution were due to infection with the same strain rather than infection with a new strain.

Abstract

Healthcare workers (HCWs) in low-incidence settings are often serially tested for latent TB infection (LTBI) with the QuantiFERON-TB Gold In-Tube (QFT) assay, which exhibits frequent conversions and reversions. The clinical impact of such variability on serial testing remains unknown. We used a microsimulation Markov model that accounts for major sources of variability to project diagnostic outcomes in a simulated North American HCW cohort. Serial testing using a single QFT with the recommended conversion cutoff (IFN-g > 0.35 IU/mL) resulted in 24.6% (95% uncertainty range, UR: 23.8-25.5) of the entire population testing false-positive over ten years. Raising the cutoff to >1.0 IU/mL or confirming initial positive results with a (presumed independent) second test reduced this false-positive percentage to 2.3% (95%UR: 2.0-2.6%) or 4.1% (95%UR: 3.7-4.5%), but also reduced the proportion of true incident infections detected within the first year of infection from 76.5% (95%UR: 66.3-84.6%) to 54.8% (95%UR: 44.6-64.5%) or 61.5% (95%UR: 51.6-70.9%), respectively. Serial QFT testing of HCWs in North America may result in tremendous over-diagnosis and over-treatment of LTBI, with nearly thirty false-positives for every true infection diagnosed. Using higher cutoffs for conversion or confirmatory tests (for initial positives) can mitigate these effects, but will also diagnose fewer true infections.

Abstract

Interferon-γ release assays (IGRAs) are blood-based tests intended for diagnosis of latent tuberculosis infection (LTBI). IGRAs offer logistical advantages and are supposed to offer improved specificity over the tuberculin skin test (TST). However, recent serial testing studies in low risk individuals have revealed higher false conversion rates with IGRAs compared with TST. Reproducibility studies have identified various sources of variability that contribute to non-reproducible results. Sources of variability can be broadly classified as pre-analytical, analytical, post-analytical, manufacturing, and immunological. In this review, we summarize known sources of variability and their impact on IGRA results. We also provide recommendations on how to minimize sources of IGRA variability.

Abstract

Clinical microbiology automation is currently limited by the lack of an in-plate culture identification system. Using an inexpensive, printed, disposable colorimetric sensor array (CSA) responsive to the volatiles emitted into plate headspace by microorganisms during growth, we report here that not only the presence but the species of bacteria growing in plate was identified before colonies are visible. In 1894 trials, 15 pathogenic bacterial species cultured on blood agar were identified with 91.0% sensitivity and 99.4% specificity within 3 hours of detection. The results indicate CSAs integrated into Petri dish lids present a novel paradigm to speciate microorganisms, well-suited to integration into automated plate handling systems.

Abstract

P. micra is an anaerobic Gram-positive cocci, and a known commensal organism of the human oral cavity and gastrointestinal tract. Although it has been classically described in association with endodontic disease and peritonsillar infection, recent reports have highlighted the role of P. micra as the primary pathogen in the setting of invasive infections. In its most recent taxonomic classification, P. micra has never been reported causing infectious endocarditis in humans. Here, we describe a 71 year-old man who developed severe native valve endocarditis complicated by aortic valvular destruction and perivalvular abscess, requiring emergent surgical intervention. Molecular sequencing enabled identification of P. micra.

Abstract

Clostridium difficile infection (CDI) is a worldwide health threat that is typically triggered by the use of broad-spectrum antibiotics, which disrupt the natural gut microbiota and allow this Gram-positive anaerobic pathogen to thrive. The increased incidence and severity of disease coupled with decreased response, high recurrence rates, and emergence of multiple antibiotic-resistant strains have created an urgent need for new therapies. We describe pharmacological targeting of the cysteine protease domain (CPD) within the C. difficile major virulence factor toxin B (TcdB). Through a targeted screen with an activity-based probe for this protease domain, we identified a number of potent CPD inhibitors, including one bioactive compound, ebselen, which is currently in human clinical trials for a clinically unrelated indication. This drug showed activity against both major virulence factors, TcdA and TcdB, in biochemical and cell-based studies. Treatment in a mouse model of CDI that closely resembles the human infection confirmed a therapeutic benefit in the form of reduced disease pathology in host tissues that correlated with inhibition of the release of the toxic glucosyltransferase domain (GTD). Our results show that this non-antibiotic drug can modulate the pathology of disease and therefore could potentially be developed as a therapeutic for the treatment of CDI.

Abstract

Interferon-gamma release assays have limited sensitivity for detecting latent tuberculosis infection. In this study, we determine if the addition of immunomodulators to the QuantiFERON-TB Gold In-Tube (QFT-GIT) increased test sensitivity without compromising specificity. We prospectively compared QFT-GIT results with and without incubation with 2 immunomodulators (lipopolysaccharide [LPS] and polyinosine-polycytidylic acid [PolyIC]) in 2 cohorts-113 culture-confirmed tuberculosis (TB) subjects in Hanoi, Vietnam, and 226 documented QFT-GIT-negative, low TB risk health care workers undergoing annual TB screening at a US academic institution. Sensitivity of the tests in TB subjects was 84.1% with the standard QFT-GIT and 85.8% and 74.3% after incubation with LPS and PolyIC, respectively. Specificity in low TB risk health care workers was 100% with the standard QFT-GIT by design and 86.7% with LPS and 63.3% with PolyIC. In conclusion, use of the 2 immunomodulators did not improve sensitivity of the QFT-GIT in TB patients and reduced specificity in low-risk health care workers.

Abstract

The diagnosis of encephalitis is particularly challenging in immunocompromised patients. We report here a case of fatal West Nile Virus encephalitis confounded by the presence of budding yeast in the CSF in a patient who had undergone heart transplantation for dilated cardiomyopathy 11 months prior to presentation of neurologic symptoms.

Abstract

Malaria is the leading identifiable cause of fever in returning travelers. Accurate Plasmodium species identification has therapy implications for P. vivax and P. ovale, which have dormant liver stages requiring primaquine. Compared to microscopy, nucleic acid tests have improved specificity for species identification and higher sensitivity for mixed infections. Here, we describe a SYBR green-based real-time PCR assay for Plasmodium species identification from whole blood, which uses a panel of reactions to detect species-specific non-18S rRNA gene targets. A pan-Plasmodium 18S rRNA target is also amplified to allow species identification or confirmation by sequencing if necessary. An evaluation of assay accuracy, performed on 76 clinical samples (56 positives using thin smear microscopy as the reference method and 20 negatives), demonstrated clinical sensitivities of 95.2% for P. falciparum (20/21 positives detected) and 100% for the Plasmodium genus (52/52), P. vivax (20/20), P. ovale (9/9), and P. malariae (6/6). The sensitivity of the P. knowlesi-specific PCR was evaluated using spiked whole blood samples (100% [10/10 detected]). The specificities of the real-time PCR primers were 94.2% for P. vivax (49/52) and 100% for P. falciparum (51/51), P. ovale (62/62), P. malariae (69/69), and P. knowlesi (52/52). Thirty-three specimens were used to test species identification by sequencing the pan-Plasmodium 18S rRNA PCR product, with correct identification in all cases. The real-time PCR assay also identified two samples with mixed P. falciparum and P. ovale infection, which was confirmed by sequencing. The assay described here can be integrated into a malaria testing algorithm in low-prevalence areas, allowing definitive Plasmodium species identification shortly after malaria diagnosis by microscopy.

Abstract

Aspergillus fumigatus (Af) and Pseudomonas aeruginosa (Pa) are leading fungal and bacterial pathogens, respectively, in many clinical situations. Relevant to this, their interface and co-existence has been studied. In some experiments in vitro, Pa products have been defined that are inhibitory to Af. In some clinical situations, both can be biofilm producers, and biofilm could alter their physiology and affect their interaction. That may be most relevant to airways in cystic fibrosis (CF), where both are often prominent residents. We have studied clinical Pa isolates from several sources for their effects on Af, including testing involving their biofilms. We show that the described inhibition of Af is related to the source and phenotype of the Pa isolate. Pa cells inhibited the growth and formation of Af biofilm from conidia, with CF isolates more inhibitory than non-CF isolates, and non-mucoid CF isolates most inhibitory. Inhibition did not require live Pa contact, as culture filtrates were also inhibitory, and again non-mucoid>mucoid CF>non-CF. Preformed Af biofilm was more resistant to Pa, and inhibition that occurred could be reproduced with filtrates. Inhibition of Af biofilm appears also dependent on bacterial growth conditions; filtrates from Pa grown as biofilm were more inhibitory than from Pa grown planktonically. The differences in Pa shown from these different sources are consistent with the extensive evolutionary Pa changes that have been described in association with chronic residence in CF airways, and may reflect adaptive changes to life in a polymicrobial environment.

Abstract

Interferon gamma (IFN-γ) release assays for latent tuberculosis infection result in a larger-than-expected number of conversions and reversions in occupational screening programs, and reproducibility of test results is a concern.Knowledge of the relative contribution and extent of the individual sources of variability (immunological, preanalytical, or analytical) could help optimize testing protocols.We performed a systematic review of studies published by October 2013 on all potential sources of variability of commercial IFN-γ release assays (QuantiFERON-TB Gold In-Tube and T-SPOT.TB). The included studies assessed test variability under identical conditions and under different conditions (the latter both overall and stratified by individual sources of variability). Linear mixed effects models were used to estimate within-subject SD.We identified a total of 26 articles, including 7 studies analyzing variability under the same conditions, 10 studies analyzing variability with repeat testing over time under different conditions, and 19 studies reporting individual sources of variability. Most data were on QuantiFERON (only three studies on T-SPOT.TB). A considerable number of conversions and reversions were seen around the manufacturer-recommended cut-point. The estimated range of variability of IFN-γ response in QuantiFERON under identical conditions was ±0.47 IU/ml (coefficient of variation, 13%) and ±0.26 IU/ml (30%) for individuals with an initial IFN-γ response in the borderline range (0.25-0.80 IU/ml). The estimated range of variability in noncontrolled settings was substantially larger (±1.4 IU/ml; 60%). Blood volume inoculated into QuantiFERON tubes and preanalytic delay were identified as key sources of variability.This systematic review shows substantial variability with repeat IFN-γ release assays testing even under identical conditions, suggesting that reversions and conversions around the existing cut-point should be interpreted with caution.

Abstract

Mycobacterium tuberculosis (Mtb) virulence is decreased by genetic deletion of the lipoprotein LprG, but the function of LprG remains unclear. We report that LprG expressed in Mtb binds to lipoglycans, such as lipoarabinomannan (LAM), that mediate Mtb immune evasion. Lipoglycan binding to LprG was dependent on both insertion of lipoglycan acyl chains into a hydrophobic pocket on LprG and a novel contribution of lipoglycan polysaccharide components outside of this pocket. An lprG null mutant (Mtb ΔlprG) had lower levels of surface-exposed LAM, revealing a novel role for LprG in determining the distribution of components in the Mtb cell envelope. Furthermore, this mutant failed to inhibit phagosome-lysosome fusion, an immune evasion strategy mediated by LAM. We propose that LprG binding to LAM facilitates its transfer from the plasma membrane into the cell envelope, increasing surface-exposed LAM, enhancing cell envelope integrity, allowing inhibition of phagosome-lysosome fusion and enhancing Mtb survival in macrophages.

Abstract

Mycobacterium tuberculosis employs various virulence strategies to subvert host immune responses in order to persist and cause disease. Interaction of M. tuberculosis with mannose receptor on macrophages via surface-exposed lipoarabinomannan (LAM) is believed to be critical for cell entry, inhibition of phagosome-lysosome fusion, and intracellular survival, but in vivo evidence is lacking. LprG, a cell envelope lipoprotein that is essential for virulence of M. tuberculosis, has been shown to bind to the acyl groups of lipoglycans but the role of LprG in LAM biosynthesis and localization remains unknown. Using an M. tuberculosis lprG mutant, we show that LprG is essential for normal surface expression of LAM and virulence of M. tuberculosis attributed to LAM. The lprG mutant had a normal quantity of LAM in the cell envelope, but its surface was altered and showed reduced expression of surface-exposed LAM. Functionally, the lprG mutant was defective for macrophage entry and inhibition of phagosome-lysosome fusion, was attenuated in macrophages, and was killed in the mouse lung with the onset of adaptive immunity. This study identifies the role of LprG in surface-exposed LAM expression and provides in vivo evidence for the essential role surface LAM plays in M. tuberculosis virulence. Findings have translational implications for therapy and vaccine development.

Abstract

Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.

Abstract

Dengue, leptospirosis, and malaria are among the most common etiologies of systemic undifferentiated febrile illness (UFI) among travelers to the developing world, and these pathogens all have the potential to cause life-threatening illness in returned travelers. The current study describes the development of an internally controlled multiplex nucleic acid amplification test for the detection of dengue virus (DENV) and Leptospira and Plasmodium species, with a specific callout for Plasmodium falciparum (referred to as the UFI assay). During analytical evaluation, the UFI assay displayed a wide dynamic range and a sensitive limit of detection for each target, including all four DENV serotypes. In a clinical evaluation including 210 previously tested samples, the sensitivities of the UFI assay were 98% for DENV (58/59 samples detected) and 100% for Leptospira and malaria (65/65 and 20/20 samples, respectively). Malaria samples included all five Plasmodium species known to cause human disease. The specificity of the UFI assay was 100% when evaluated with a panel of 66 negative clinical samples. Furthermore, no amplification was observed when extracted nucleic acids from related pathogens were tested. Compared with whole-blood samples, the UFI assay remained positive for Plasmodium in 11 plasma samples from patients with malaria (parasitemia levels of 0.0037 to 3.4%). The syndrome-based design of the UFI assay, combined with the sensitivities of the component tests, represents a significant improvement over the individual diagnostic tests available for these pathogens.

Abstract

Antimicrobial drug resistance in treatment of urinary tract infection (UTI) continues to rise worldwide. To examine contributions of physician prescribing patterns to fluoroquinolone (ciprofloxacin, CP) resistance, we examined Escherichia coli (E. coli) resistance patterns in urinary cultures. Since CP usage is limited in children, we compared CP resistance trends in adults and children to those of more commonly used trimethoprim-sulfamethoxazole (TMP-SMX) and nitrofurantoin (NF). Our data show that although the general pediatric population has lower resistance to ciprofloxacin, resistance levels are rising with increased usage. While NF susceptibility is historically stable, TMP-SMX resistance is slightly higher in children compared to adults. In both adults and children, antimicrobial resistance patterns vary according to clinical practice site, with ambulatory urology patients showing the highest resistance. This suggests that physician's prescribing patterns contribute to antimicrobial resistance.

Abstract

Sepsis is a medical emergency demanding early diagnosis and tailored antimicrobial therapy. Every hour of delay in initiating effective therapy measurably increases patient mortality. Blood culture is currently the reference standard for detecting bloodstream infection, a multistep process which may take one to several days. Here, we report a novel paradigm for earlier detection and the simultaneous identification of pathogens in spiked blood cultures by means of a metabolomic "fingerprint" of the volatile mixture outgassed by the organisms. The colorimetric sensor array provided significantly faster detection of positive blood cultures than a conventional blood culture system (12.1 h versus 14.9 h, P < 0.001) while allowing for the identification of 18 bacterial species with 91.9% overall accuracy within 2 h of growth detection. The colorimetric sensor array also allowed for discrimination between unrelated strains of methicillin-resistant Staphylococcus aureus, indicating that the metabolomic fingerprint has the potential to track nosocomial transmissions. Altogether, the colorimetric sensor array is a promising tool that offers a new paradigm for diagnosing bloodstream infections.

Abstract

Clostridium difficile infection (CDI) is the most common cause of infectious diarrhea in health care settings, and for patients presumed to have CDI, their isolation while awaiting laboratory results is costly. Newer rapid tests for CDI may reduce this burden, but the economic consequences of different testing algorithms remain unexplored. We used decision analysis from the hospital perspective to compare multiple CDI testing algorithms for adult inpatients with suspected CDI, assuming patient management according to laboratory results. CDI testing strategies included combinations of on-demand PCR (odPCR), batch PCR, lateral-flow diagnostics, plate-reader enzyme immunoassay, and direct tissue culture cytotoxicity. In the reference scenario, algorithms incorporating rapid testing were cost-effective relative to nonrapid algorithms. For every 10,000 symptomatic adults, relative to a strategy of treating nobody, lateral-flow glutamate dehydrogenase (GDH)/odPCR generated 831 true-positive results and cost $1,600 per additional true-positive case treated. Stand-alone odPCR was more effective and more expensive, identifying 174 additional true-positive cases at $6,900 per additional case treated. All other testing strategies were dominated by (i.e., more costly and less effective than) stand-alone odPCR or odPCR preceded by lateral-flow screening. A cost-benefit analysis (including estimated costs of missed cases) favored stand-alone odPCR in most settings but favored odPCR preceded by lateral-flow testing if a missed CDI case resulted in less than $5,000 of extended hospital stay costs and <2 transmissions, if lateral-flow GDH diagnostic sensitivity was >93%, or if the symptomatic carrier proportion among the toxigenic culture-positive cases was >80%. These results can aid guideline developers and laboratory directors who are considering rapid testing algorithms for diagnosing CDI.

Abstract

Identification and treatment of latent tuberculosis infection (LTBI) can substantially reduce the risk of developing active disease. However, there is no diagnostic gold standard for LTBI. Two tests are available for identification of LTBI: the tuberculin skin test (TST) and the gamma interferon (IFN-γ) release assay (IGRA). Evidence suggests that both TST and IGRA are acceptable but imperfect tests. They represent indirect markers of Mycobacterium tuberculosis exposure and indicate a cellular immune response to M. tuberculosis. Neither test can accurately differentiate between LTBI and active TB, distinguish reactivation from reinfection, or resolve the various stages within the spectrum of M. tuberculosis infection. Both TST and IGRA have reduced sensitivity in immunocompromised patients and have low predictive value for progression to active TB. To maximize the positive predictive value of existing tests, LTBI screening should be reserved for those who are at sufficiently high risk of progressing to disease. Such high-risk individuals may be identifiable by using multivariable risk prediction models that incorporate test results with risk factors and using serial testing to resolve underlying phenotypes. In the longer term, basic research is necessary to identify highly predictive biomarkers.

Abstract

This case illustrates skin lesions in a traveler staying in a hotel bed infested with tics. Although infestation of hotels with bedbugs belonging to the Cimex genus is a growing problem worldwide, tick infestation has never been reported before.

Abstract

We report the second pediatric case of New Delhi metallo-beta-lactamase (NDM-1)-producing Enterobacteriaceae in the United States in a girl from India who presented to a teaching hospital in Northern California with cystitis due to NDM-1-producing E. coli and K. pneumoniae. Laboratory methods included various phenotypic antimicrobial susceptibility testing assays, as well as PCR assays for carbapenemase-encoding genes. Laboratory challenges included a false negative modified Hodge test and reversion of carbapenem resistance in the E. coli strain. The limited number of effective antimicrobial agents and the lack of pediatric-specific safety and efficacy data for these drugs presented significant therapeutic challenges.

Abstract

North American occupational health programs that switched from the tuberculin skin test (TST) to IFN-γ release assays for latent tuberculosis screening are reporting challenges with interpretation of serial testing results in healthcare workers (HCWs). However, limited data exist on the reproducibility of serial IFN-γ release assay results in low-risk HCWs.To evaluate the short-term reproducibility of QuantiFERON-TB Gold In-Tube (QFT) in a large cohort of HCWs and to define a QFT cutoff yielding a conversion rate equivalent to historical TST rates.We retrospectively evaluated the QFT results from HCWs with two or more QFT tests performed between June 2008 and July 2010 at an academic institution. Outcome measures were proportions of reproducibility, quantitative results, and conversion rates with alternate QFT cutoffs.A total of 9,153 HCWs with two or more QFT tests were included in the analysis. Of 8,227 individuals with a negative result, 4.4% (n = 361) converted their QFT result over 2 years. A total of 261 (72.3%) of the HCWs with conversions underwent repeat short-term testing after the first positive result with 64.8% reverting (n = 169). An IFN-γ cutoff of 5.3 IU/ml or higher (manufacturer's cutoff is ≥0.35 IU/ml) yielded a conversion rate of 0.4%, equal to our institution's historical TST conversion rate.The manufacturer's definition of QFT conversion results in an inflated conversion rate that is incompatible with our low-risk setting. A significantly higher QFT cutoff value is needed to match the historical TST conversion rate. Nonreproducible conversions in most converters suggested false-positive results.

Abstract

Rapid and accurate detection of Shiga-toxin producing E. coli of all serotypes from patients with diarrhea is critical for medical management and for prevention of ongoing transmissions. In this prospective study, we assessed the performance of a multiplex, real-time PCR assay targeting stx1 and stx2 for detection of O157 and non-O157 Shiga-toxin producing E. coli from diarrheal stool samples enriched in GN broth. We show that the assay is 100% sensitive (95% confidence interval (CI), 89.1% to 100%) and 98.5% specific (95% CI, 90.6% to 99.9%), based on a panel of 40 known STEC-positive and 65 known negative specimens. During a two-year post-validation period, the assay detected a greater number of positive samples from patients in Northern California compared to culture and PCR testing performed at a public health reference laboratory, with a positive predictive value of 95.6% (95% CI, 87.6% to 99.1%). Serotyping data showed an incidence rate of 51.2% for non-O157 STEC strains with 5.8% (1/17) of patients with non-O157 strains and 42.9% (6/14) with O157 strains (P=0.03) developing hemolytic uremic syndrome. The findings from this study underscore the recommendations of the CDC for laboratories to test all diarrheal stool samples from patients with acute community-acquired diarrhea for non-O157 STEC in addition to O157 serotype using a sensitive assay. Additionally, a survey of 17 clinical laboratories in Northern California demonstrated that nearly 50% do not screen all stool specimens for the presence of Shiga toxins, indicating that many clinical microbiology laboratories still do not routinely screen all stool specimens for the presence of Shiga toxins recommended in the 2009 CDC guidelines.

Abstract

Tuberculosis (TB) continues to be a public health emergency, compounded by the lack of adequate diagnostic testing in many regions of the world. New advances in the molecular detection of Mycobacterium tuberculosis, including faster and simpler nucleic acid amplification tests, have resulted in rapid and cost-effective methods to diagnose TB and test for drug resistance. Ongoing research on biomarkers for TB infection may lead to new tests for blood, urine, breath, and sputum. Sustained investment in the development and dissemination of diagnostic tests for TB is critical for increasing TB case finding, placing patients on appropriate treatment, and reducing transmission.

Abstract

Objectives: To describe and discuss the utility and potential pitfalls of ribosomal RNA locus sequencing for direct identification of invasive fungi from fresh and formalin-fixed, paraffin-embedded specimens. Methods: DNA was extracted from fresh and formalin-fixed, paraffin-embedded tissue and subjected to real-time polymerase chain reaction (PCR) targeting ITS2 and D2 regions of fungal ribosomal RNA locus. Cycle sequencing was performed on PCR products, and the identity of sequences was determined using a public database. Results: Four clinical cases of invasive fungal infection are presented to illustrate the utility of DNA sequencing for determining etiology when microbiological culture is negative, for shortening the time to identification of slow-growing fungi, for guiding antifungal therapy, and for shedding light on the pathogenesis of disseminated fungal infection. Conclusions: Fungal ribosomal RNA locus sequencing from fresh or formalin-fixed, paraffin-embedded specimens is a powerful tool for rapid and accurate diagnosis of patients with culture-negative or uncultured invasive mycosis.

Abstract

Previous work showed that cell imprinting in a polydimethylsiloxane (PDMS) film produced artificial receptors to cells by template-assisted rearrangement of functional groups on the surface of the polymer thin film which facilitated cell capture in the polymer surface indentations by size, shape, and most importantly chemical recognition. We report here that inactivation of cells by treatment with formaldehyde (4%), or glutaraldehyde (2%), or a combination of the two leads to markedly improved capture selectivity (a factor of 3) when cells to be analyzed are inactivated in the same manner. The enhanced capture efficiency compared to living cells results from two factors: (1) rigidification of the cell surface through crosslinking of amine groups by the aldehyde; and (2) elimination of chemicals excreted from living cells which interfere with the fidelity of the cell imprinting process. Moreover, cell inactivation has the advantage of removing biohazard risks associated with working with virulent bacteria. These results are demonstrated using different strains of mycobacterium tuberculosis.

Abstract

Current tools available to study the molecular epidemiology of tuberculosis do not provide information about the directionality and sequence of transmission for tuberculosis cases occurring over a short period of time, such as during an outbreak. Recently, whole genome sequencing has been used to study molecular epidemiology of Mycobacterium tuberculosis over short time periods.To describe the microevolution of M. tuberculosis during an outbreak caused by one drug-susceptible strain. METHOD AND MEASUREMENTS: We included 9 patients with tuberculosis diagnosed during a period of 22 months, from a population-based study of the molecular epidemiology in San Francisco. Whole genome sequencing was performed using Illumina's sequencing by synthesis technology. A custom program written in Python was used to determine single nucleotide polymorphisms which were confirmed by PCR product Sanger sequencing.We obtained an average of 95.7% (94.1-96.9%) coverage for each isolate and an average fold read depth of 73 (1 to 250). We found 7 single nucleotide polymorphisms among the 9 isolates. The single nucleotide polymorphisms data confirmed all except one known epidemiological link. The outbreak strain resulted in 5 bacterial variants originating from the index case A1 with 0-2 mutations per transmission event that resulted in a secondary case.Whole genome sequencing analysis from a recent outbreak of tuberculosis enabled us to identify microevolutionary events observable during transmission, to determine 0-2 single nucleotide polymorphisms per transmission event that resulted in a secondary case, and to identify new epidemiologic links in the chain of transmission.

Abstract

Reports regarding the incidence and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) in rhinosinusitis (RS) are limited. This study was designed to identify epidemiology and trends of MRSA incidence and antimicrobial resistance in the sinonasal cavities.This is a retrospective case series. All intranasal/sinus cultures obtained by otolaryngologists at Stanford over a 20-year period (1990-2010) were retrospectively reviewed by mining the microbiology database. Nested searches were then made for all S. aureus and MRSA cultures. Patterns of incidence and changes in antibiotic susceptibilities were tabulated and statistical analysis was performed.Our search retrieved 10,387 positive intranasal culture samples, with S. aureus found in 800 (7.7%), and MRSA comprising 110 (1.06%) of this subset. Between the years of 1990 and 1999, only 2/112 (1.7%) of S. aureus-positive nasal cultures were positive for MRSA, with a sharp rise in incidence to 86/606 (14.2%) from 2000 to 2005, and to 22/82, 26.8% from 2006 to 2010. On a percent basis, using logistic regression modeling, this represents a statistically significant increasing trend (p < 0.0001) for MRSA sinusitis. However, over the 20-year interval studied, the patterns of antibiotic resistance among MRSA remained unaltered, especially with regard to trimethoprim-sulfamethoxazole and vancomycin.S. aureus and MRSA isolates from intranasal cultures, which were essentially absent before the year 2000, became significantly more common earlier this decade. These data show the increased role of MRSA in sinusitis. MRSA antibiotic susceptibilities have remained, however, largely stable during this time period.

Abstract

Activation of innate immunity via pathogen recognition receptors (PRR) modulates adaptive immune responses. PRR ligands are being exploited as vaccine adjuvants and as therapeutics, but their utility in diagnostics has not been explored. Interferon-gamma (IFN-γ) release assays (IGRAs) are functional T cell assays used to diagnose latent tuberculosis infection (LTBI); however, novel approaches are needed to improve their sensitivity.In vitro immunomodulation of a whole blood IGRA (QuantiFERON®-TB GOLD In-Tube) with Toll-like receptor agonists poly(I:C), LPS, and imiquimod was performed on blood from subjects with LTBI and negative controls.In vitro immunomodulation significantly enhanced the response of T cells stimulated with M. tuberculosis antigens from subjects with LTBI but not from uninfected controls. Immunomodulation of IGRA revealed T cell responses in subjects with LTBI whose T cells otherwise do not respond to in vitro stimulation with antigens alone. Similar to their in vivo functions, addition of poly(I:C) and LPS to whole blood induced secretion of inflammatory cytokines and IFN-α and enhanced the surface expression of antigen presenting and costimulatory molecules on antigen presenting cells.In vitro immunomodulation of whole blood IGRA may be an effective strategy for enhancing the sensitivity of T cells for diagnosis of LTBI.

Abstract

We investigated a sudden increase in the rate of positive QuantiFERON-TB Gold In-Tube results from 10% to 31% at a U.S. academic institution. Direct comparison of the TB antigen tubes with tubes from a different lot number identified that a potential problem with the TB antigen vials in a certain tube lot was the likely cause of the elevated positive rate. The underlying defect remains unknown. This finding warrants refinement of quality control programs by the manufacturer and users.

Abstract

Microscopic diagnosis and species identification of Plasmodium in areas of nonendemicity provide a robust method for malaria diagnosis but are technically challenging. A prospective study was conducted to measure the performance of BinaxNOW compared to microscopy (the gold standard) in a U.S. teaching hospital. Overall, BinaxNOW was 84.2% sensitive and 99.8% specific. Excluding patients on antimalarial therapy, the sensitivity was 92.9%. Importantly, BinaxNOW initially misclassified a case of Plasmodium falciparum malaria as non-falciparum. These results support the judicious use of BinaxNOW in screening of individuals suspected of having malaria in areas of nonendemicity.

Abstract

The sensitivity of QuantiFERON-TB GOLD In-Tube was measured in 104 subjects with recent (≤2 years) and remote Mycobacterium tuberculosis infection using tuberculin skin test conversion as the reference standard. The sensitivity was not significantly different between the 2 groups (33% versus 20%, P = 0.3). This finding suggests interferon-γ release assays may not be more sensitive for diagnosis of recent than remote infection. Longitudinal studies are needed to validate this finding.

Abstract

The rapid and accurate diagnosis of tuberculosis (TB) in children and extrapulmonary TB in adults continues to be a challenge. In this study, we determined the lower limit of detection (LOD) of the GeneXpert MTB/RIF assay with nonrespiratory specimens and investigated the utility of flotation procedures for concentrating the bacilli. Clinical specimens (9 cerebrospinal fluid [CSF], 13 gastric aspirate, 8 tissue, and 17 stool) were spiked with single-celled Mycobacterium tuberculosis, and the LOD of the GeneXpert assay was determined. Flotation studies were conducted with sucrose and NaCl, and the cycle thresholds of the MTB/RIF assay were compared between treated and untreated samples. There was no significant difference between the LODs of the GeneXpert assay with saline solution (median, 33 CFU/ml) and CSF (median, 25 CFU/ml) (P > 0.05) or gastric aspirate samples (median, 58 CFU/ml) (P > 0.05). The LOD with spiked tissue (median, 1,525 CFU/ml) and stool samples (median, 6,800 CFU/ml) was significantly elevated compared to that determined with saline solution (P ≤ 0.05 and ≤ 0.0005, respectively). Flotation studies with sucrose or NaCl did not consistently result in lowered cycle thresholds in stool or gastric aspirates, but a cycle reduction of >10 was achieved in two of the three pooled CSF samples. Unlike the results seen with tissue and stool samples, there was no significant PCR inhibition in the MTB/RIF assay with CSF and gastric aspirates. Although preconcentration of CSF samples with sucrose and NaCl may enhance detection of M. tuberculosis by PCR, further advances are needed to concentrate the bacilli and eliminate PCR inhibitors in paucibacillary nonrespiratory samples.

Abstract

Cryptococcus species are known agents of opportunistic infections in healthy and immunocompromised hosts. Here we describe the first case of Cryptococcus uzbekistanensis causing bone marrow infection in an elderly Asian man with undiagnosed T cell lymphoma presenting with fever of unknown origin, pancytopenia, and exposure to chicken manure.

Abstract

The emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) producing acquired carbapenemases have created a global public health crisis. In the United States, CRE producing the Klebsiella pneumoniae carbapenemase (KPC) are increasingly common and are endemic in some regions. Metallo-β-lactamase (MBL)-producing CRE have recently been reported in the United States among patients who received medical care in countries where such organisms are common. Here, we describe three carbapenem-resistant K. pneumoniae isolates recovered from pediatric patients at a single U.S. health care facility, none of whom had a history of international travel. The isolates were resistant to carbapenems but susceptible to aztreonam, trimethoprim-sulfamethoxazole, and fluoroquinolones. The three isolates were closely related to each other by pulsed-field gel electrophoresis and contained a common plasmid. PCR and sequence analysis confirmed that these isolates produce IMP-4, an MBL carbapenemase not previously published as present among Enterobacteriaceae in the United States.

Abstract

Adaptive immunity to Mycobacterium tuberculosis controls progressive bacterial growth and disease but does not eradicate infection. Among CD4+ T cells in the lungs of M. tuberculosis-infected mice, we observed that few produced IFN-γ without ex vivo restimulation. Therefore, we hypothesized that one mechanism whereby M. tuberculosis avoids elimination is by limiting activation of CD4+ effector T cells at the site of infection in the lungs. To test this hypothesis, we adoptively transferred Th1-polarized CD4+ effector T cells specific for M. tuberculosis Ag85B peptide 25 (P25TCRTh1 cells), which trafficked to the lungs of infected mice and exhibited antigen-dependent IFN-γ production. During the early phase of infection, ∼10% of P25TCRTh1 cells produced IFN-γ in vivo; this declined to <1% as infection progressed to chronic phase. Bacterial downregulation of fbpB (encoding Ag85B) contributed to the decrease in effector T cell activation in the lungs, as a strain of M. tuberculosis engineered to express fbpB in the chronic phase stimulated P25TCRTh1 effector cells at higher frequencies in vivo, and this resulted in CD4+ T cell-dependent reduction of lung bacterial burdens and prolonged survival of mice. Administration of synthetic peptide 25 alone also increased activation of endogenous antigen-specific effector cells and reduced the bacterial burden in the lungs without apparent host toxicity. These results indicate that CD4+ effector T cells are activated at suboptimal frequencies in tuberculosis, and that increasing effector T cell activation in the lungs by providing one or more epitope peptides may be a successful strategy for TB therapy.

Abstract

Interferon-release assays (IGRAs) represent advances in tuberculosis immunology and evolutionary biology. IGRAs were designed to replace tuberculin skin test (TST) for the diagnosis of latent tuberculosis infection because of their logistical advantages and enhanced specificity over TST. Although IGRAs and TST have been useful in epidemiologic studies, they lack the sensitivity and reproducibility normally expected from diagnostic tests in clinical practice. In this review, we present an overview of the current recommendations and knowledge in the field and discuss practical approaches in areas of uncertainty related to discordant IGRA results.

Abstract

Phaeoacremonium parasiticum is an environmental fungus usually associated with subcutaneous infections. We report the first documented case of central nervous system involvement with brain abscess formation in a patient with chronic granulomatous disease and review the literature on Phaeoacremonium parasiticum infections.

Abstract

Polyfluorophores built on a DNA scaffold (ODFs) were synthesized and tested for fluorescence responses to the volatiles from M. tuberculosis, E. coli and P. putida in closed Petri dishes. Two sensors in a pattern-based response could distinguish the bacterial strains accurately, suggesting the use of ODFs in rapid identification of infectious agents.

Abstract

Patients with diarrhea, defined as loose or watery stool, and two or more Clostridium difficile tcdB PCR tests within 14 days of each other were investigated. Repeat PCR for 293 patients with a prior negative result yielded negative results in 396 (97.5%) of 406 tests. Ten new positives were detected, including one false positive. Repeat PCR within 7 days appears rarely useful, except for patients with evidence of a new infection.

Abstract

In vitro gamma interferon release assays (IGRAs) are increasingly used as an alternative to the traditional tuberculin skin test for the diagnosis of latent Mycobacterium tuberculosis infection. Evaluation of the QuantiFERON-TB Gold in-tube assay (QFT-IT) prior to large-scale implementation at the Stanford Hospital and Clinics for a health care worker screening program revealed a critical preanalytical factor affecting the results. We found that incubation delay significantly increased the frequency of indeterminate results. In this study, QFT-IT was performed with samples from healthy volunteers, and replicate tubes were incubated at 37 degrees C either immediately or after a delay at room temperature for 6 and 12 h. No indeterminate results (0/41) were seen when the assay was performed with immediate incubation. Incubation delays of 6 and 12 h yielded indeterminate results at rates of 10% (2/20) (P = 0.10) and 17.1% (7/41) (P = 0.01), respectively. The increased rate of indeterminate results was due to a decrease in the mean values for the mitogen-nil tubes when incubation was delayed for 6 h (P = 0.004) and 12 h (P < 0.001). The rates of concordance of positive or negative results obtained following immediate incubation and following 6- and 12-h delays were 77.8% (14/18) and 79.4% (27/34), respectively. Subsequent implementation of the immediate incubation procedure in our screening program for 14,830 health care workers yielded an indeterminate result rate of 0.36% over a period of 12 months, a significant improvement over the reported rates of 5 to 40% for QFT-IT. We conclude that immediate incubation of QFT-IT tubes is an effective way to minimize indeterminate results. The effect of incubation delay on the accuracy of QFT-IT remains to be determined.

Abstract

The Mycobacterium chelonae-Mycobacterium abscessus group (MCAG) is the most common cause of infections because of rapidly growing mycobacteria. Rapid identification of MCAG to the species level is essential for choosing empiric antibiotic treatment and for public health measures. In this study, we compared the performance of a single-tube multiplex, real-time polymerase chain reaction (PCR) assay to 3 biochemical tests for species-level identification of 46 MCAG isolates. We show that real-time PCR provides the most accurate results for rapid species-level identification of MCAG.

Abstract

Real-time PCR can rapidly identify Mycobacterium tuberculosis in paraffin-embedded tissue in the absence of microbiological culture. In a comparison of single-copy and multicopy PCR targets in 70 tissue samples, the sensitivities were 26% and 54%, respectively, with 100% specificity. Sensitivity was 75% for newer samples and was not decreased for acid-fast bacillus (AFB) stain-negative specimens.

Abstract

A representative case in which a polymicrobial infection involving Fusobacterium nucleatum, Actinomyces israelii and Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans was initially diagnosed as malignancy in an edentulous patient. Additional history obtained after the nature of the syndrome was elucidated revealed that he had had his two remaining teeth extracted four months prior to this episode.

Abstract

Nucleic acid amplification tests (NAATs) have revolutionized infectious disease diagnosis, allowing for the rapid and sensitive identification of pathogens in clinical specimens. Real-time PCR testing for the mecA gene (mecA PCR), which confers methicillin resistance in staphylococci, has the added potential to reduce antibiotic usage, improve clinical outcomes, lower health care costs, and avoid emergence of drug resistance. A retrospective study was performed to identify patients infected with methicillin-sensitive staphylococcal isolates who were receiving vancomycin treatment when susceptibility results became available. Vancomycin treatment and length of hospitalization were compared in these patients for a 6-month period before and after implementation of mecA PCR. Among 65 and 94 patients identified before and after mecA PCR, respectively, vancomycin usage (measured in days on therapy) declined from a median of 3 days (range, 1 to 44 days) in the pre-PCR period to 1 day (range, 0 to 18 days) in the post-PCR period (P < 0.0001). In total, 38.5% (25/65) of patients were switched to beta-lactam therapy in the pre-PCR period, compared to 61.7% (58/94) in the post-PCR period (P = 0.004). Patient hospitalization days also declined from a median of 8 days (range, 1 to 47 days) in the pre-PCR period to 5 days (range, 0 to 42 days) in the post-PCR period (P = 0.03). Real-time PCR testing for mecA is an effective tool for reducing vancomycin usage and length of stay of hospitalized patients infected with methicillin-sensitive staphylococci. In the face of ever-rising health care expenditures in the United States, these findings have important implications for improving outcomes and decreasing costs.

Abstract

We report a case of 2009 influenza A(H1N1) virus infection in which virus was detected predominantly in specimens from the lower respiratory tract but was absent or at very low levels in nasopharyngeal swab samples. This presentation suggests that, in certain hosts or for particular variants of 2009 A(H1N1) virus, the lower respiratory tract may be the preferred site of infection.

Abstract

Drug-resistant strains of Mycobacterium tuberculosis are increasing worldwide and pose a major threat to global health. However, it remains unsettled whether drug-resistant mutants are fixed in the bacterial population or if they would revert in the absence of drug pressure.To document the occurrence of isoniazid (INH) reversion in a patient with multidrug-resistant tuberculosis (TB) and investigate its association with fitness cost.Genotypic and phenotypic assays were used to characterize the reversion of INH resistance in isolates from a patient with pulmonary TB. The pre-reversion katG mutation was reconstructed in a pan-susceptible laboratory strain (H37Rv DeltakatG::katG W300G) and tested for susceptibility to INH and oxidative stress.Genotyping and drug susceptibility testing showed that an isogenic strain of M. tuberculosis reverted from an INH-resistant to a susceptible phenotype in the absence of INH therapy. The genotypic basis of this reversion was mapped to the katG codon 300 which reverted from GGG (glycine, G) to a wild-type codon, TGG (tryptophan, W). The H37Rv DeltakatG::katG W300G mutant was resistant to INH, but also showed a deficiency in coping with oxidative stress.This study confirms that, in the absence of INH pressure, some INH-resistant mutants will revert to a drug-susceptible phenotype. This finding may have broader implications for INH-resistant strains and for the clinically useful lifespan of INH.

Abstract

Staphylococcus lugdunensis is an aggressive, virulent member of the coagulase-negative staphylococci (CoNS) that is responsible for severe, rapidly progressive skin and soft tissue infections and native valve endocarditis. To facilitate prompt identification and appropriate therapy, we describe here a rapid and robust multiplex real-time PCR assay that is able to definitively distinguish S. lugdunensis from other staphylococci. Using melting curve analysis, the assay also identifies Staphylococcus aureus and CoNS other than S. lugdunensis and determines MecA-dependent resistance to methicillin (meticillin). When applied to a panel of well-characterized staphylococcal reference strains, as well as 165 clinical isolates previously identified by conventional methods, the assay was both sensitive and specific for S. lugdunensis, correctly identifying the reference strain and all 47 S. lugdunensis isolates without inappropriate amplification of other staphylococci. Furthermore, rapid biochemical identification using the WEE-TAB system to detect ornithine decarboxylase activity was found to be unsuitable as an alternative to PCR identification, displaying just 31% sensitivity and 77% specificity when tested on a subset (90 isolates) of the clinical strains. We therefore propose that this simple, accurate PCR approach will allow for the routine and timely identification of S. lugdunensis in the clinical microbiology laboratory.

Abstract

Malachite green, a synthetic antimicrobial dye, has been used for over 50 years in mycobacterial culture medium to inhibit the growth of contaminants. The molecular basis of mycobacterial resistance to malachite green is unknown, although the presence of malachite green-reducing enzymes in the cell envelope has been suggested. The objective of this study was to investigate the role of lipoproteins in resistance of Mycobacterium tuberculosis to malachite green. The replication of an M. tuberculosis lipoprotein signal peptidase II (lspA) mutant (DeltalspA::lspAmut) on Middlebrook agar with and without 1 mg/liter malachite green was investigated. The lspA mutant was also compared with wild-type M. tuberculosis in the decolorization rate of malachite green and sensitivity to sodium dodecyl sulfate (SDS) detergent and first-line antituberculosis drugs. The lspA mutant has a 10(4)-fold reduction in CFU-forming efficiency on Middlebrook agar with malachite green. Malachite green is decolorized faster in the presence of the lspA mutant than wild-type bacteria. The lspA mutant is hypersensitive to SDS detergent and shows increased sensitivity to first-line antituberculosis drugs. In summary, lipoprotein processing by LspA is essential for resistance of M. tuberculosis to malachite green. A cell wall permeability defect is likely responsible for the hypersensitivity of lspA mutant to malachite green.

Abstract

Disseminated infection due to nontuberculous Mycobacterium (NTM) species is rare in pediatrics. Here we report 6 infections affecting 5 patients at a single institution in an immunocompromised population of pediatric oncology and stem cell transplant recipients. The patients presented within a 1-year period with catheter-associated bacteremia. New pulmonary nodules were noted in 4 of the 5 patients. All of the infections were due to rapidly growing NTM. Patients were successfully treated with removal of the infected catheter and combination antibiotic therapy. There are currently no consensus guidelines for treatment of NTM infections in this population, and a therapeutic approach is presented here.

Abstract

Blood agar is used for the identification and antibiotic susceptibility testing of many bacterial pathogens. In the developing world, microbiologists use human blood agar because of the high cost and inhospitable conditions for raising wool sheep or horses to supply blood. Many pathogens either fail to grow entirely or exhibit morphologies and hemolytic patterns on human blood agar that confound colony recognition. Furthermore, human blood can be hazardous to handle due to HIV and hepatitis. This study investigated whether blood from hair sheep, a hardy, low-maintenance variety of sheep adapted for hot climates, was suitable for routine clinical microbiology studies.Hair sheep blood obtained by jugular venipuncture was anticoagulated by either manual defibrination or collection in human blood bank bags containing citrate-phosphate-dextrose. Trypticase soy 5% blood agar was made from both forms of hair sheep blood and commercial defibrinated wool sheep blood. Growth characteristics, colony morphologies, and hemolytic patterns of selected human pathogens, including several streptococcal species, were evaluated. Specialized identification tests, including CAMP test, reverse CAMP test, and satellite colony formation with Haemophilus influenzae and Abiotrophia defectiva were also performed. Mueller-Hinton blood agar plates prepared from the three blood types were compared in antibiotic susceptibility tests by disk diffusion and E-test.The results of all studies showed that blood agar prepared from citrated hair sheep blood is suitable for microbiological tests used in routine identification and susceptibility profiling of human pathogens. The validation of citrated hair sheep blood eliminates the labor-intensive and equipment-requiring process of manual defibrination. Use of hair sheep blood, in lieu of human blood currently used by many developing world laboratories and as an alternative to cost-prohibitive commercial sheep blood, offers the opportunity to dramatically improve the safety and accuracy of laboratory diagnosis of pathogenic bacteria in resource-poor countries.

Abstract

Klebsiella pneumoniae serogroups displaying the hypermucoviscosity phenotype are associated with a distinct clinical syndrome characterized by liver abscesses, bacteraemia and metastatic lesions. We describe here what we believe to be the first reported case of hypermucoviscous K. pneumoniae causing a superficial Bartholin's abscess in the absence of systemic involvement.

Abstract

The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n = 96), MAC (n = 97), MCAG (n = 68), and M. mucogenicum (n = 9) and 95% and 100%, respectively, for MFG (n = 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.

Abstract

Despite the advantages of providing an early presumptive diagnosis, fungal classification by histopathology can be difficult and may lead to diagnostic error. To assess the accuracy of histologic diagnosis of fungal infections vs culture ("gold standard"), we performed a 10-year retrospective review at our institution. Of the 47 of 338 positive mold and yeast cultures with concurrent surgical pathology evaluation without known history of a fungal infection, 37 (79%) were correctly identified based on morphologic features in histologic and/or cytologic specimens. The 10 discrepant diagnoses (21%) included misidentification of septate and nonseptate hyphal organisms and yeast forms. Errors resulted from morphologic mimics, use of inappropriate terminology, and incomplete knowledge in mycology. The accuracy did not correlate with preceding antifungal therapy (P = .14) or use of special stains (P = .34) and was not operator-dependent. Among 8 discrepancies with clinical follow-up available, 2 potential adverse clinical consequences resulted. While histopathologic identification of fungi in tissue sections and cytologic preparations is prone to error, implementation of a standardized reporting format should improve diagnostic accuracy and prevent adverse outcomes.

Abstract

The species identification of members of the Mycobacterium tuberculosis complex is critical to the timely initiation of both appropriate antibiotic therapy and proper public health control measures. However, the current commercially available molecular assays identify mycobacteria only to the complex level and are unable to differentiate M. tuberculosis from the closely related M. bovis and M. bovis BCG. We describe here a rapid and robust two-step, multiplex, real-time PCR assay based on genomic deletions to definitively identify M. tuberculosis, M. bovis, M. bovis BCG, and other members of the complex. When tested against a panel of well-characterized mycobacterial reference strains, the assay was both sensitive and specific, correctly identifying all strains. We applied this assay to 60 clinical isolates previously identified as M. tuberculosis complex and found 57 M. tuberculosis isolates and 3 M. bovis BCG isolates from patients who had received intravesical BCG. Furthermore, analysis of 15 clinical specimens previously identified as M. bovis by spoligotyping revealed an isolate of M. tuberculosis that had been misidentified. We propose that this assay will allow the routine identification of M. tuberculosis complex members in the clinical laboratory.

Abstract

The onset of the adaptive immune response to Mycobacterium tuberculosis is delayed compared with that of other infections or immunization, and allows the bacterial population in the lungs to expand markedly during the preimmune phase of infection. We used adoptive transfer of M. tuberculosis Ag85B-specific CD4(+) T cells to determine that the delayed adaptive response is caused by a delay in initial activation of CD4(+) T cells, which occurs earliest in the local lung-draining mediastinal lymph node. We also found that initial activation of Ag85B-specific T cells depends on production of antigen by bacteria in the lymph node, despite the presence of 100-fold more bacteria in the lungs. Although dendritic cells have been found to transport M. tuberculosis from the lungs to the local lymph node, airway administration of LPS did not accelerate transport of bacteria to the lymph node and did not accelerate activation of Ag85B-specific T cells. These results indicate that delayed initial activation of CD4(+) T cells in tuberculosis is caused by the presence of the bacteria in a compartment that cannot be mobilized from the lungs to the lymph node, where initial T cell activation occurs.

Abstract

In a prospective study conducted by laboratory technologists in a diagnostic laboratory in Cape Town, South Africa, a semi-automated phage-based antibiotic susceptibility assay was implemented and the performance of the luciferase reporter mycobacteriophage (LRP) system for susceptibility testing of clinical Mycobacterium tuberculosis complex (MTC) isolates against rifampin and isoniazid was evaluated. Two hundred consecutive clinical MGIT cultures of MTC species were included in this study. Antibiotic susceptibility assays were set up manually for the LRP and BACTEC radiometric systems (BACTEC) and read in a plate luminometer and the BACTEC 460 instrument, respectively. Discrepant susceptibility results were resolved by the conventional agar proportion method. Of the 200 secondary cultures prepared for this study, 9 (4.5%) were lost to contamination (LRP 4, BACTEC 1, both 4). All of the remaining 191 cultures underwent susceptibility testing by both methods and the overall agreement between the LRP and BACTEC was 98.4% (rifampin 100%; isoniazid 96.9%). Of the 6 discrepant cultures tested by the agar proportion method, 2 gave results in agreement with the LRP. The sensitivity of the LRP for detection of drug-resistant isolates was 100% for both rifampin (n=9) and isoniazid (n=12). The median turnaround time for susceptibility testing was 2 days with the LRP and 9 days with BACTEC. In conclusion, the semi-automated LRP-based assay offers a rapid and practical approach for accurate susceptibility testing of M. tuberculosis cultures in diagnostic laboratories with limited financial resources, but with competent technologists.

Abstract

The objective of this study was to investigate the antimicrobial activity and specificity of globomycin, an inhibitor of lipoprotein signal peptidase II (LspA), against Mycobacterium tuberculosis.The mycobactericidal and mycobacteriostatic activity of globomycin was determined by optical density and cfu plating. The specificity of globomycin was determined by western immunoblotting using anti-MPT83 antibody.Globomycin is mycobactericidal at concentrations>or=40 mg/L. However, at 80 mg/L, the processing of the lipoprotein MPT-83 is unaffected and growth-inhibitory effect of globomycin is unchanged in an lspA null mutant (DeltalspA::lspAmut) lacking the putative drug target.Globomycin kills M. tuberculosis through a mechanism that is independent of LspA.

Abstract

Tuberculosis kills nearly 2 million people annually, and current approaches to tuberculosis control are expensive, have limited efficacy, and are vulnerable to being overcome by extensively drug-resistant strains of Mycobacterium tuberculosis. Determination of the genome sequence of M. tuberculosis has revolutionized tuberculosis research, contributed to major advances in the understanding of the evolution and pathogenesis of M. tuberculosis, and facilitated development of new diagnostic tests with increased specificity for tuberculosis. In this review, we describe some of the major progress in tuberculosis research that has resulted from knowledge of the genome sequence and note some of the problems that remain unsolved.

Abstract

Mycobacterium tuberculosis is a highly successful human pathogen, with approximately 2x10(9) individuals infected globally. To understand the responses of M. tuberculosis to the in vivo environment, we studied the in vivo regulation of M. tuberculosis genes whose M. marinum homologs are induced in chronically infected frog tissues. The expression of 16S rRNA was shown to remain constant in M. tuberculosis under in vivo and in vitro conditions and therefore could be used for internal normalization in quantitative reverse transcription-PCR assays. We found whiB3, a putative transcriptional regulator implicated in mediating tissue damage, to be maximally induced at 2 weeks postinfection in the lungs of wild-type and immunodeficient (gamma interferon receptor-/-, Rag1-/-, and tumor necrosis factor alpha-/-) mice. At later time points in wild-type mice, whiB3 induction was decreased and gradually declined over the course of infection. In immunodeficient mice, whiB3 induction declined rapidly and was completely abolished in moribund animals. whiB3 was also found to be induced in naïve bone marrow-derived macrophages after 6 h of infection. whiB3 expression in vivo and in vitro was found to be inversely correlated with bacterial density. These results indicate that M. tuberculosis regulates the expression of whiB3 in response to environmental signals present in vivo and are consistent with a model of regulation by quorum sensing.

Abstract

Mycobacterium tuberculosis is a highly successful pathogen that can persist and cause disease despite an immune response. One potential mechanism for resisting elimination is by inhibiting the action of IFN-gamma. We have previously shown that live M. tuberculosis inhibits selected macrophage responses to IFN-gamma, and that purified M. tuberculosis 19-kDa lipoprotein inhibits induction of selected IFN-gamma-responsive genes through a TLR2-dependent pathway, whereas peptidoglycan inhibits responses to IFN-gamma by a TLR2-independent pathway. To determine the relative contribution of lipoproteins to the inhibition of responses to IFN-gamma, we deleted the M. tuberculosis gene (lspA) that encodes lipoprotein signal peptidase. This revealed that M. tuberculosis lipoprotein processing is indispensable for stimulation of TLR2 reporter cells, but that the lspA mutant inhibits macrophage responses to IFN-gamma to the same extent as wild-type bacteria. Macrophages lacking TLR2 are more resistant to inhibition by either strain of M. tuberculosis, suggesting that nonlipoprotein TLR2 agonists contribute to inhibition. Indeed, we found that phosphatidylinositol mannan from M. tuberculosis inhibits macrophage responses to IFN-gamma. M. tuberculosis inhibition of responses to IFN-gamma requires new protein synthesis, indicating that a late effect of innate immune stimulation is the inhibition of responses to IFN-gamma. These results establish that M. tuberculosis possesses multiple mechanisms of inhibiting responses to IFN-gamma.

Abstract

In a prospective study conducted in a diagnostic laboratory in Mexico City, luciferase reporter mycobacteriophages (LRPs) were evaluated for their utility and performance in identification and antibiotic-susceptibility testing of Mycobacterium tuberculosis complex (MTC) isolates from MGIT-960 cultures. Eighty-four consecutive MGIT cultures recovered from 54 patients were included in this study. The LRPs confirmed mycobacterial growth in 79 (94 %) of 84 MGIT cultures. Failure to confirm growth was due to low inoculum (n = 1) or growth with non-tuberculous mycobacteria (n = 4). The median time to confirmation of MGIT cultures was 1 day (range 1-55). Confirmed cultures were identified with p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP), a selective inhibitor of MTC species, and results obtained with LRPs were compared with those obtained by BACTEC-460. The sensitivity and specificity of the LRP NAP test were respectively 97 and 100 %, and the median turnaround time for identification was 3 days with both methods. The accuracy and speed of the LRPs for susceptibility testing with rifampicin, streptomycin, isoniazid and ethambutol were compared with BACTEC-460 and discrepant results were tested by the conventional agar proportion method. In total, 72 MTC cultures were tested. The overall agreement between the LRPs and BACTEC-460 was 98.6 %. Four isolates (5.6 %) were falsely identified as ethambutol-resistant. The median turnaround time for susceptibility testing was 3 days (range 3-57) with the LRPs and 9 days (range 7-29) with BACTEC-460. LRPs offer an accurate and rapid approach for identification and susceptibility testing of M. tuberculosis from MGIT-960 cultures.

Abstract

Rapid diagnosis of drug-resistant M.tuberculosis (Mtb) is desirable worldwide. We (i) describe a new luciferase reporter phage (LRP), phAE142 for this purpose; (ii) compare it to the automated MGIT 960 for time-to-detection of Mtb in clinical specimens; and (iii) evaluate its use for species confirmation and antibiotic susceptibility testing(AST) of Mtb. Twenty sputum samples were inoculated for testing by LRP, or by MGIT 960. After "positives" were identified by either method, the LRP was used for confirmation of Mtb complex (TBC) and for AST. The LRP method proved comparably efficient to MGIT 960 at detecting Mtb. Using an antibiotic uniquely inhibiting TBC with LRP provided species assignment, concurrently with AST, in a median of 3 days, with a sensitivity of 97%. Overall agreement in susceptibility results was 96%. Reliable susceptibility results and identification of TBC can be completed in a median of 12 days (range 8 to 16d) with LRP applied to sputum samples.

Abstract

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p-nitro-alpha-acetylamino-beta-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

Abstract

Defensins constitute a family of 3- to 4-kDa antimicrobial peptides that are stored in the cytoplasmic granules of neutrophils, some macrophages, and intestinal Paneth cells. We have assessed defensin gene expression during myeloid differentiation by first characterizing cDNAs for each of the four known rat neutrophil defensins (RatNP 1-4). The cDNA sequences revealed that the peptides are synthesized as 87-94 amino acid precursors, each containing signal, pro-, and mature peptide segments. RatNP-3 and -4 mRNAs, but not those for RatNP-1 and -2 or other myeloid defensins, contained unique polypurine tracts located close to the termination codon in the 3' untranslated region. By using cDNA probes and/or riboprobes, we evaluated defensin transcript levels in a variety of tissues and in the full spectrum of neutrophil precursors. By in situ hybridization, defensin mRNAs were localized to neutrophil precursors in the bone marrow, with the highest mRNA levels occurring in promyelocytes and somewhat lower signals occurring in myeloblasts and myelocytes. Defensin mRNAs were not detectable in bands or mature neutrophils, nor at significant levels in tissues other than bone marrow. The accumulation of defensin protein in bone marrow cells was assessed by immunohistochemical staining with anti-RatNP-1 Ab. RatNP 1-4 mRNAs and protein levels were then correlated for each stage of neutrophilic differentiation to reveal the maturational profile of myeloid defensin gene expression in the rat.